| Rhododendron seniavinii is a semi-evergreen shrub of the genus Ericaceae,in the family Rhododendron.It is not only the main plant raw material of R.seniavinii syrup,a kind of traditional Chinese medicine for the treatment of acute or chronic bronchitis,but also has specific effects on cardiovascular and cerebrovascular,liver and anti-tumor.With the increasing human demand,the wild resources of R.seniavinii are greatly reduced.R.seniavinii has a long reproductive growth cycle,and seeds are small and difficult to collect.Therefore,the cutting breeding technology as a kind of high efficiency,low cost,fast propagation speed,and maintaining the excellent characteristics of the variety that can be used to solve the key problem.This study used cuttings with semi-lignified of 1year age of R.seniavinii as research materials.It used three factors（type,concentration,and soaking time）with three levels（L9,34）and orthogonal test design to observe the rooting index of cuttings.On this basis,the morphogenesis,physiological indexes and transcriptome of the rooting process with the highest rooting rate were correlated and analyzed,and further insight into the influencing factors and rooting mechanism of cuttings in R.seniavinii.The findings were as follows:1.Each Rooting index of R.seniavinii.The optimal combination for rooting rate was the treatment with ABT(200mg L-1)and 2 hours of soaking time（G8）.The optimal combination for average adventitious root number was the treatment with ABT(200mg L-1)and 4 hours of soaking time.The optimal combination for average adventitious root length was the treatment with IBA(400mg L-1)and 2 hours of soaking time.The best combination for the longest adventitious roots was the treatment with NAA(100mg L-1)and 2 hours of soaking time.The best combination for rooting index was the treatment with ABT(200mg L-1)and 2 hours of soaking time（G8）.The optimal combination for rooting rate was the treatment with NAA(400mg L-1)and 4 hours of soaking time.2.Morphogenesis of adventitious roots of R.seniavinii.The adventitious root formation was divided into three developmental stages,namely T1（primitive stage）,T2（initial stage）and T3（expression stage）in R.seniavinii.The shape of phenotype was not significantly different between the T1 and T2 stages in R.seniavinii.The root primordia were differentiated and grew outward to form adventitious roots in the T3 stage.The number and length of adventitious roots formed by cutting slips in G8 were significantly higher than those of CK.Observed from the microscopic structure,the layers of longitudinal paraffin sections were well-defined in order of pith,xylem,vascular cambium,phloem,cortex and epidermis,and no adventitious root primordium were found in the T1stage.Partial cortical thin-walled cells divide into thin-walled cells leaded to adventitious root primordium in the T2 stage.The anterior end of adventitious root primordium develops to a root crown,and the posterior end of adventitious root primordia was continues broken through the epidermis through the grow of cell in elongation zone in the T3 stage.For the microscopic structure,there was no significant difference between G8and CK.Therefore,the rooting mode was cutaneous rooting and its root primordium was induced rooting of R.seniavinii.3.Nutrients relationships during rooting in R.seniavinii cuttings.In both G8 and CK,the soluble sugar and soluble protein in the base of cuttings decreased first in T2 period and increased in T3 period.In the spike leaves,the soluble sugar and soluble protein of G8 increased in T2 and decreased in T3.The soluble sugar and soluble protein of CK showed a continuous increase.The variation range of nutrients in G8cuttings was significantly greater than that in CK.The used of growth regulators promoted the flow of nutrients in the spike to the base of the spike.The used of growth regulators facilitates the completion of dedifferentiation and redifferentiation of parenchyma cells in the spike,shortening the time of rooting and improving the rooting rate of the spike.4.Relationship of oxidative enzyme activities in the rooting process of R.seniavinii cuttings.During adventitious root formation,IAAO activity was decreasing first and then increasing,while PPO and POD activities were decreasing.The PPO activity of G8 treatment showed a rebound in T3 period,but remained lower than in T1 period.G8treatment significantly reduced the PPO and POD activities and reduced the IAAO activity.Therefore,higher IAAO activity and lower PPO and POD activity were more favorable for the formation and growth of adventitious roots of R.seniavinii cuttings.5.Endogenous hormone relationships in the rooting process of cuttings.The IAA content at the base of the cuttings was down-up in G8,and was continuously increased of CK.ABA content was rising-falling in G8,and was falling-rising in CK.GA content was down-up in G8,and was up-down in CK.ZR content in G8 was showed a continuous increase,and that in CK was showed a decrease-increase.All spike leaves showed higher IAA,ABA,GA,and ZR contents in G8 than in CK,and indicated that the G8 facilitates the accumulation of these four endogenous hormones in the spike leaves,thus promoting early rooting of the spike.6.Mechanism of adventitious root formation from cuttings.The63,761 differentially expressed genes were identified in spike leaves,and which 39,685 genes were upregulated and,24,076 genes were down-regulated.The 175,602 differentially expressed genes were identified at the plug’s base,of which 93,038 were upregulated,and82,564 were down-regulated.The 278 differential genes at the base of the spike were significantly enriched in the T1 vs T2 and T2 vs T3phytohormone signaling pathways,and G8 upregulated significantly more differential genes than CK.The function of DEGs indicated that G8was beneficial in increasing differential gene expression in R.seniavinii,which shortened the formation time of adventitious roots and increased the rooting rate and the number of roots.The WOX family genes of R.seniavinii cuttings were identified and analyzed for expression.Rs WOX5,Rs WOX6,Rs WOX7,Rs WOX8,Rs WOX9,Rs WOX10 and Rs WOX12 were more closely related to Arabidopsis thaliana.By the expression analysis,we hypothesized that the Rs WOX5,Rs WOX6 and Rs WOX12 were to be important for adventitious root formation of R.seniavinii.7.Differential gene expression related to aerial rooting and ground root.The transcriptome analysis of aerial roots and ground roots showed 4,579 differentially expressed genes,of which 2,391 showed up-regulation and 2,188 showed down-regulation.Most differential genes are related to functions such as transformation and oxidase activity and are mainly enriched in metabolic pathways and ribosomes.Analysis of differential gene expression revealed that the expression of Asd3 was significantly higher in aerial roots than in ground roots,which was presumed essential in forming aerial roots.Ad1,Ad2,Ad3,Ad4,Ad5,Asd1&Asd2 have high expression,and it is presumed that these genes have essential roles in adventitious root growth.In conclusions,the G8 treatment significantly improved the rooting index and rooting rate of R.seniavinii.After sensing trauma and growth substance signals,spikes import extra-membrane signals into the cell via transmembrane conversion,and endogenous hormone genes associated with root primordium formation are subsequently activated.The four hormone signaling networks in vivo further amplify the signal to promote up-regulated expression of rooting genes.Nutrient flow in the spike to the base of the spike,high IAAO and low PPO,formation of POD activity,and promotion of adventitious root primordium formation in R.seniavinii.The cells then continue to divide and gradually differentiate into meristematic tissues with different functional domains,forming mature root tips and penetrating the epidermis at the base of the spike to form adventitious roots.This may be the mechanism of rooting cuttings for the propagation in the cortex of R.seniavinii. |
